Matrix metalloproteinase-9 and -2 enhance the ligand sensitivity of photoreceptor cyclic nucleotide-gated channels

Photoreceptor cyclic nucleotide-gated (CNG) channels are the principal ion channels responsible for transduction of the light-induced change in cGMP concentration into an electrical signal. The ligand sensitivity of photoreceptor CNG channels is subject to regulation by intracellular signaling effectors, including calcium-calmodulin, tyrosine kinases and phosphoinositides. Little is known, however, about regulation of channel activity by modification to extracellular regions of CNG channel subunits. Extracellular proteases MMP9 and -2 are present in the interphotoreceptor matrix adjacent to photoreceptor outer segments. Given that MMPs have been implicated in retinal dysfunction and degeneration, we hypothesized that MMP activity may alter the functional properties of photoreceptor CNG channels. For heterologously expressed rod and cone CNG channels, extracellular exposure to MMPs dramatically increased the apparent affinity for cGMP and the efficacy of cAMP. These changes to ligand sensitivity were not prevented by destabilization of the actin cytoskeleton or by disruption of integrin mediated cell adhesion, but could be attenuated by inhibition of MMP catalytic activity. MMP-mediated gating changes exhibited saturable kinetic properties consistent with enzymatic processing of the CNG channels. In addition, exposure to MMPs decreased the abundance of full-length expressed CNGA3 subunits, with a concomitant increase in putative degradation products. Similar gating effects and apparent proteolysis were observed also for native rod photoreceptor CNG channels. Furthermore, constitutive apparent proteolysis of retinal CNGA1 and retinal MMP9 levels were both elevated in aged mice compared with young mice. Together, these results provide evidence that MMP-mediated proteolysis can regulate the ligand sensitivity of CNG channels.     

Matrix metalloproteinase-9 and -2 enhance the ligand sensitivity of photoreceptor cyclic nucleotide-gated channels

Photoreceptor cyclic nucleotide-gated (CNG) channels are the principal ion channels responsible for transduction of the light-induced change in cGMP concentration into an electrical signal. The ligand sensitivity of photoreceptor CNG channels is subject to regulation by intracellular signaling effectors, including calcium-calmodulin, tyrosine kinases and phosphoinositides. Little is known, however, about regulation of channel activity by modification to extracellular regions of CNG channel subunits. Extracellular proteases MMP9 and -2 are present in the interphotoreceptor matrix adjacent to photoreceptor outer segments. Given that MMPs have been implicated in retinal dysfunction and degeneration, we hypothesized that MMP activity may alter the functional properties of photoreceptor CNG channels. For heterologously expressed rod and cone CNG channels, extracellular exposure to MMPs dramatically increased the apparent affinity for cGMP and the efficacy of cAMP. These changes to ligand sensitivity were not prevented by destabilization of the actin cytoskeleton or by disruption of integrin mediated cell adhesion, but could be attenuated by inhibition of MMP catalytic activity. MMP-mediated gating changes exhibited saturable kinetic properties consistent with enzymatic processing of the CNG channels. In addition, exposure to MMPs decreased the abundance of full-length expressed CNGA3 subunits, with a concomitant increase in putative degradation products. Similar gating effects and apparent proteolysis were observed also for native rod photoreceptor CNG channels. Furthermore, constitutive apparent proteolysis of retinal CNGA1 and retinal MMP9 levels were both elevated in aged mice compared with young mice. Together, these results provide evidence that MMP-mediated proteolysis can regulate the ligand sensitivity of CNG channels.     

Purification and cloning of toxins from elapid venoms that target cyclic nucleotide-gated ion channels

In 1999, we purified pseudechetoxin (PsTx), the first peptide toxin known to block cyclic nucleotide-gated (CNG) ion channels, from the venom of Pseudechis australis [Brown, R. L., Haley, T. L., West, K. A., and Crabb, J. W. (1999) Proc. Natl. Acad. Sci. U.S.A. 96, 754-759]. Here we report the cloning of the cDNA encoding PsTx, as well as the discovery and cloning of pseudecin, a homologous toxin from the venom of Pseudechis porphyriacus. The mature proteins are 211 and 210 amino acids in length, and the amino acid sequences are 96.7% identical, differing in only seven residues. The purified toxins were applied to outside-out patches excised from Xenopus oocytes expressing CNG channels composed of the rod CNGA1 or olfactory CNGA2 channel subunits. Surprisingly, these patch-clamp studies revealed a 30-fold difference in affinity between PsTx and pseudecin for channels composed of CNGA2 subunits. The apparent K(i) of PsTx was 15 nM, while the affinity of pseudecin was 460 nM. The difference in affinities for the CNGA1 subunit from rod photoreceptors was less pronounced, but the affinity of PsTx was 70 nM, compared with 1000 nM for pseudecin. This difference in affinity may be instructive as we attempt to identify the regions of the toxins that contact CNG channels. As the only known protein blockers of CNG channels, these toxins promise to be valuable tools to study the structure of the external face of these channels.     

Pseudechetoxin binds to the pore turret of cyclic nucleotide-gated ion channels

Peptide toxins are invaluable tools for studying the structure and physiology of ion channels. Pseudechetoxin (PsTx) is the first known peptide toxin that targets cyclic nucleotide-gated (CNG) ion channels, which play a critical role in sensory transduction in the visual and olfactory systems. PsTx inhibited channel currents at low nM concentrations when applied to the extracellular face of membrane patches expressing olfactory CNGA2 subunits. Surprisingly, 500 nM PsTx did not inhibit currents through channels formed by the CNGA3 subunit from cone photoreceptors. We have exploited this difference to identify the PsTx-binding site on the extracellular face of CNG channels. Studies using chimeric channels revealed that transplantation of the pore domain from CNGA2 was sufficient to confer high affinity PsTx binding upon a CNGA3 background. To further define the binding site, reciprocal mutations were made at 10 nonidentical amino acid residues in this region. We found that two residues in CNGA2, D316 and Y321, were essential for high-affinity inhibition by PsTx. Furthermore, replacement of both residues was required to confer high-affinity PsTx inhibition upon CNGA3. Several other residues, including E325, also form favorable interactions with PsTx. In the CNGA2-E325K mutant, PsTx affinity was reduced by approximately 5-fold to 120 nM. An electrostatic interaction with D316 does not appear to be the primary determinant of PsTx affinity, as modification of the D316C mutant with a negatively charged methanethiosulfonate reagent did not restore high affinity inhibition. The residues involved in PsTx binding are found within the pore turret and helix, in similar positions to residues that form the receptor for pore-blocking toxins in voltage-gated potassium channels. Furthermore, biophysical properties of PsTx block, including an unfavorable interaction with permeant ions, also suggest that it acts as a pore blocker. In summary, PsTx seems to occlude the entrance to the pore by forming high-affinity contacts with the pore turret, which may be larger than that found in the KcsA structure.     

Functional activity of photoreceptor cyclic nucleotide-gated channels is dependent on the integrity of cholesterol- and sphingolipid-enriched membrane domains

Rod and cone photoreceptor cyclic nucleotide-gated (CNG) channels play pivotal roles in phototransduction. This work investigates the functional significance of photoreceptor CNG channel association with membrane microdomains enriched in raft lipids, cholesterol and sphingolipids. The primary subunits of cone and rod CNG channels, CNGA3 and CNGA1, respectively, were heterologously expressed in HEK 293 cells, and channel activity was determined by ratiometric measurement of [Ca (2+)] i in response to cyclic guanosine monophosphate (cGMP) stimulation. CNGA3 was found to be largely insoluble following Triton X-100 extraction and cofractionationed with biochemically isolated membrane domains enriched in caveolin-1. Cofractionation of both natively expressed CNGA3 and CNGB1 (the modulatory subunit of the rod CNG channel) with the low buoyant density, caveolin-1-enriched membranes was also confirmed in mouse retinas. The functional significance of this association was established by the observed negative effects of depletion of raft lipids on the channel activity. Treatment with the cholesterol depleting agent, methyl-beta-cyclodextrin (MCD), significantly inhibited CNGA3 and CNGA1 activation in response to cGMP stimulation. MCD treatment lowered cellular cholesterol levels by approximately 45% without altering fatty acid composition, suggesting that the inhibition of channel activity by MCD treatment is not due to perturbation of other membrane lipids. Treatment with the sphingolipid biosynthesis inhibitor myriocin resulted in impaired activation and cytosolic redistribution of CNGA3, suggesting that the integrity of the membrane domains is critical for the channel cellular processing and plasma membrane localization. This study demonstrates the association of photoreceptor CNG channels with membrane domains enriched in raft lipids and indicates, for the first time, that raft lipids modulate the plasma membrane localization and functional activity of photoreceptor CNG channels.     

Dynamics of Ca2+-calmodulin-dependent inhibition of rod cyclic nucleotide-gated channels measured by patch-clamp fluorometry

Cyclic nucleotide-gated (CNG) ion channels mediate cellular responses to sensory stimuli. In vertebrate photoreceptors, CNG channels respond to the light-induced decrease in cGMP by closing an ion-conducting pore that is permeable to cations, including Ca(2+) ions. Rod CNG channels are directly inhibited by Ca(2+)-calmodulin (Ca(2+)/CaM), but the physiological role of this modulation is unknown. Native rod CNG channels comprise three CNGA1 subunits and one CNGB1 subunit. The single CNGB1 subunit confers several key properties on heteromeric channels, including Ca(2+)/CaM-dependent modulation. The molecular basis for Ca(2+)/CaM inhibition of rod CNG channels has been proposed to involve the binding of Ca(2+)/CaM to a site in the NH(2)-terminal region of the CNGB1 subunit, which disrupts an interaction between the NH(2)-terminal region of CNGB1 and the COOH-terminal region of CNGA1. Here, we test this mechanism for Ca(2+)/CaM-dependent inhibition of CNGA1/CNGB1 channels by simultaneously monitoring protein interactions with fluorescence spectroscopy and channel function with patch-clamp recording. Our results show that Ca(2+)/CaM binds directly to CNG channels, and that binding is the rate-limiting step for channel inhibition. Further, we show that the NH(2)- and COOH-terminal regions of CNGB1 and CNGA1 subunits, respectively, are in close proximity, and that Ca(2+)/CaM binding causes a relative rearrangement or separation of these regions. This motion occurs with the same time course as channel inhibition, consistent with the notion that rearrangement of the NH(2)- and COOH-terminal regions underlies Ca(2+)/CaM-dependent inhibition.     

Role of the S4-S5 linker in CNG channel activation

Cyclic nucleotide-gated (CNG) channels mediate sensory signal transduction in retinal and olfactory cells. The channels are activated by the binding of cyclic nucleotides to a cyclic nucleotide-binding domain (CNBD) in the C-terminus that is located at the intracellular side. The molecular events translating the ligand binding to the pore opening are still unknown. We investigated the role of the S4-S5 linker in the activation process by quantifying its interaction with other intracellular regions. To this end, we constructed chimeric channels in which the N-terminus, the S4-S5 linker, the C-linker, and the CNBD of the retinal CNGA1 subunit were systematically replaced by the respective regions of the olfactory CNGA2 subunit. Macroscopic concentration-response relations were analyzed, yielding the apparent affinity to cGMP and the Hill coefficient. The degree of functional coupling of intracellular regions in the activation gating was determined by thermodynamic double-mutant cycle analysis. We observed that all four intracellular regions, including the relatively short S4-S5 linker, are involved in controlling the apparent affinity of the channel to cGMP and, moreover, in determining the degree of cooperativity between the subunits, as derived from the Hill coefficient. The interaction energies reveal an interaction of the S4-S5 linker with both the N-terminus and the C-linker, but no interaction with the CNBD.     

Distinct binding properties distinguish LQ-type calmodulin-binding domains in cyclic nucleotide-gated channels

Cyclic nucleotide-gated (CNG) channels operate as transduction channels in photoreceptors and olfactory receptor neurons. Direct binding of cGMP or cAMP opens these channels which conduct a mixture of monovalent cations and Ca(2+). Upon activation, CNG channels generate intracellular Ca(2+) signals that play pivotal roles in the transduction cascades of the visual and olfactory systems. Channel activity is controlled by negative feedback mechanisms that involve Ca(2+)-calmodulin, for which all CNG channels possess binding sites. Here we compare the binding properties of the two LQ-type calmodulin binding sites, both of which are thought to be involved in channel regulation. They reside on the isoforms CNGB1 and CNGA4. The CNGB1 subunit is present in rod photoreceptors and olfactory receptor neurons. The CNGA4 subunit is only expressed in olfactory receptor neurons, and there are conflicting results as to its role in calmodulin-mediated feedback inhibition. We examined the interaction of Ca(2+)-calmodulin with two recombinant proteins that encompass either of the two LQ sites. Comparing binding properties, we found that the LQ site of CNGB1 binds Ca(2+)-calmodulin at 10-fold lower Ca(2+) levels than the LQ site of CNGA4. Our data provide biochemical evidence against a contribution of CNGA4 to feedback inhibition. In accordance with previous work on photoreceptor CNG channels, our results indicate that feedback control is the exclusive role of the B-subunits in photoreceptors and olfactory receptor neurons.     

Calcium/calmodulin modulation of olfactory and rod cyclic nucleotide-gated ion channels

Cyclic nucleotide-gated (CNG) ion channels mediate sensory transduction in olfactory sensory neurons and retinal photoreceptor cells. In these systems, internal calcium/calmodulin (Ca2+/CaM) inhibits CNG channels, thereby having a putative role in sensory adaptation. Functional differences in Ca2+/CaM-dependent inhibition depend on the different subunit composition of olfactory and rod CNG channels. Recent evidence shows that three subunit types (CNGA2, CNGA4, and CNGB1b) make up native olfactory CNG channels and account for the fast inhibition of native channels by Ca2+/CaM. In contrast, two subunit types (CNGA1 and CNGB1) appear sufficient to mirror the native properties of rod CNG channels, including the inhibition by Ca2+/CaM. Within CNG channel tetramers, specific subunit interactions also mediate Ca2+/CaM-dependent inhibition. In olfactory CNGA2 channels, Ca2+/CaM binds to an N-terminal region and disrupts an interaction between the N- and C-terminal regions, causing inhibition. Ca2+/CaM also binds the N-terminal region of CNGB1 subunits and disrupts an intersubunit, N- and C-terminal interaction between CNGB1 and CNGA1 subunits in rod channels. However, the precise N- and C-terminal regions that form these interactions in olfactory channels are different from those in rod channels. Here, we will review recent advances in understanding the subunit composition and the mechanisms and roles for Ca2+/CaM-dependent inhibition in olfactory and rod CNG channels.     

Functional consequences of progressive cone dystrophy-associated mutations in the human cone photoreceptor cyclic nucleotide-gated channel CNGA3 subunit

Progressive cone dystrophies are a genetically heterogeneous group of disorders characterized by early deterioration of visual acuity and color vision, together with psychophysical and electrophysiological evidence of abnormal cone function and cone degeneration. Recently, three mutations in the gene encoding the CNGA3 subunit of cone photoreceptor cyclic nucleotide-gated (CNG) channels have been linked to progressive cone dystrophy in humans. To investigate the functional consequences of these mutations, we expressed mutant human CNGA3 subunits in Xenopus oocytes, alone or together with human CNGB3, and studied these channels using patch-clamp recording. Compared with wild-type channels, homomeric and heteromeric channels containing CNGA3-N471S or CNGA3-R563H subunits exhibited an increase in apparent affinity for cGMP and an increase in the relative agonist efficacy of cAMP compared with cGMP. In contrast, R277C subunits did not form functional homomeric or heteromeric channels. Cell surface expression levels, determined using confocal microscopy of green fluorescent protein-tagged subunits and patch-clamp recording, were significantly reduced for both R563H and R277C but unchanged for N471S. Overall, these results suggest that the plasma membrane localization and gating properties of cone CNG channels are altered by progressive cone dystrophy-associated mutations, providing evidence that supports the pathogenicity of these mutations. phosphodiesterase     

Dequalinium: a novel,high-affinity blocker of CNGA1 channels

Cyclic nucleotide-gated (CNG) channels have been shown to be blocked by diltiazem, tetracaine, polyamines, toxins, divalent cations, and other compounds. Dequalinium is an organic divalent cation which suppresses the rat small conductance Ca(2+)-activated K(+) channel 2 (rSK2) and the activity of protein kinase C. In this study, we have tested the ability of dequalinium to block CNGA1 channels and heteromeric CNGA1+CNGB1 channels. When applied to the intracellular side of inside-out excised patches from Xenopus oocytes, dequalinium blocks CNGA1 channels with a K(1/2) approximately 190 nM and CNGA1+CNGB1 channels with a K(1/2) approximately 385 nM, at 0 mV. This block occurs in a state-independent fashion, and is voltage dependent with a zdelta approximately 1. Our data also demonstrate that dequalinium interacts with the permeant ion probably because it occupies a binding site in the ion conducting pathway. Dequalinium applied to the extracellular surface also produced block, but with a voltage dependence that suggests it crosses the membrane to block from the inside. We also show that at the single-channel level, dequalinium is a slow blocker that does not change the unitary conductance of CNGA1 channels. Thus, dequalinium should be a useful tool for studying permeation and gating properties of CNG channels.     

15-Deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2) mediates repression of TNF-alpha by decreasing levels of acetylated histone H3 and H4 at its promoter

Thirty-nine missense mutations, which had been identified in rod monochromacy or related disorders, in the CNGA3 subunit of cone photoreceptor cGMP-gated channels were analyzed. HEK293 cells were transfected with cDNA of the human CNGA3 subunit harboring each of these mutations in an expression vector. Patch-clamp recordings demonstrated that 32 of the 39 mutants did not show cGMP-activated current, suggesting that these 32 mutations cause a loss of function of the channels. From the remaining 7 mutants that showed cGMP-activated current, two mutations in the cyclic nucleotide-binding domain, T565M or E593K, were further studied. The half-maximal activating concentration (K(1/2)) for cGMP in the homomeric CNGA3-T565M channels (160microM) was 17.8-fold higher than that of the homomeric wild-type CNGA3 channels (9.0microM). Conversely, the K(1/2) for cGMP in the homomeric CNGA3-E593K channels (3.0microM) was 3-fold lower than that of the homomeric wild-type CNGA3 channels. These results suggest that the T565M and E593K mutations alter the apparent affinity for cGMP of the channels to cause cone dysfunction, resulting in rod monochromacy.     

Two structural components in CNGA3 support regulation of cone CNG channels by phosphoinositides

Cyclic nucleotide-gated (CNG) channels in retinal photoreceptors play a crucial role in vertebrate phototransduction. The ligand sensitivity of photoreceptor CNG channels is adjusted during adaptation and in response to paracrine signals, but the mechanisms involved in channel regulation are only partly understood. Heteromeric cone CNGA3 (A3) + CNGB3 (B3) channels are inhibited by membrane phosphoinositides (PIP_n), including phosphatidylinositol 3,4,5-triphosphate (PIP₃) and phosphatidylinositol 4,5-bisphosphate (PIP₂), demonstrating a decrease in apparent affinity for cyclic guanosine monophosphate (cGMP).Unlike homomeric A1 or A2 channels, A3-only channels paradoxically did not show a decrease in apparent affinity for cGMP after PIP_n application. However, PIP_n induced an ∼2.5-fold increase in cAMP efficacy for A3 channels. The PIP_n-dependent change in cAMP efficacy was abolished by mutations in the C-terminal region (R643Q/R646Q) or by truncation distal to the cyclic nucleotide-binding domain (613X). In addition, A3-613X unmasked a threefold decrease in apparent cGMP affinity with PIP_n application to homomeric channels, and this effect was dependent on conserved arginines within the N-terminal region of A3. Together, these results indicate that regulation of A3 subunits by phosphoinositides exhibits two separable components, which depend on structural elements within the N- and C-terminal regions, respectively. Furthermore, both N and C regulatory modules in A3 supported PIP_n regulation of heteromeric A3+B3 channels. B3 subunits were not sufficient to confer PIP_n sensitivity to heteromeric channels formed with PIP_n-insensitive A subunits. Finally, channels formed by mixtures of PIP_n-insensitive A3 subunits, having complementary mutations in N- and/or C-terminal regions, restored PIP_n regulation, implying that intersubunit N–C interactions help control the phosphoinositide sensitivity of cone CNG channels.     

Achromatopsia-associated mutation in the human cone photoreceptor cyclic nucleotide-gated channel CNGB3 subunit alters the ligand sensitivity and pore properties of heteromeric channels

Cone photoreceptor cyclic nucleotide-gated (CNG) channels are thought to form by assembly of two different subunit types, CNGA3 and CNGB3. Recently, mutations in the gene encoding the CNGB3 subunit have been linked to achromatopsia in humans. Here we describe the functional consequences of two achromatopsia-associated mutations in human CNGB3 (hCNGB3). Co-expression in Xenopus oocytes of human CNGA3 (hCNGA3) subunits with hCNGB3 subunits containing an achromatopsia-associated mutation in the S6 transmembrane domain (S435F) generated functional heteromeric channels that exhibited an increase in apparent affinity for both cAMP and cGMP compared with wild type heteromeric channels. In contrast, co-expression of a presumptive null mutation of hCNGB3 (T383f.s.Delta C) with hCNGA3 produced channels with properties indistinguishable from homomeric hCNGA3 channels. The effect of hCNGB3 S435F subunits on cell-surface expression of green fluorescent protein-tagged hCNGA3 subunits and of non-tagged hCNGA3 subunits on surface expression of green fluorescent protein-hCNGB3 S435F subunits were similar to those observed for wild type hCNGB3 subunits, suggesting that the mutation does not grossly disturb subunit assembly or plasma membrane targeting. The S435F mutation was also found to produce changes in the pore properties of the channel, including decreased single channel conductance and decreased sensitivity to block by l-cis-diltiazem. Overall, these results suggest that the functional properties of cone CNG channels may be altered in patients with the S435F mutation, providing evidence supporting the pathogenicity of this mutation in humans. Thus, achromatopsia may arise from a disturbance of cone CNG channel gating and permeation or from the absence of functional CNGB3 subunits.     

Distinct structural determinants of efficacy and sensitivity in the ligand-binding domain of cyclic nucleotide-gated channels

Cyclic nucleotide-gated (CNG) channels open in response to direct binding of cyclic nucleotide messengers. Every subunit in a tetrameric CNG channel contains a cytoplasmic ligand-binding domain (BD) that includes a beta-roll (flanked by short helices) and a single C-terminal helix called the C-helix that was previously found to control efficacy (maximal open probability) and selectivity for cGMP versus cAMP. We constructed a series of chimeric CNG channel subunits, each containing a distinct BD sequence (chosen from among six phylogenetically divergent isoforms) fused to an invariant non-BD sequence. We assayed these "BD substitution" chimeras as homomeric CNG channels in Xenopus oo-cytes to compare their functions and found that the most efficient activation by both cAMP and cGMP derived from the BD of the catfish CNGA4 olfactory modulatory subunit (fCNGA4). We then tested the effects of replacing subregions of the bovine CNGA1 BD with corresponding fCNGA4 sequence and hence identified parts of the fCNGA4 BD producing efficient activation. For instance, replacing either the "hinge" that connects the roll and C-helix subdomains or the BD sequence N-terminal to the hinge greatly enhanced cAMP efficacy. Replacing the "loop-beta 8" region (the C-terminal end of the beta-roll) improved agonist sensitivity for cGMP selectively over cAMP. Our results thus identify multiple BD elements outside the C-helix that control selective ligand interaction and channel gating steps by distinct mechanisms. This suggests that the purine ring of the cyclic nucleotide may interact with both the beta-roll and the C-helix at different points in the mechanism.     

Noncatalytic Inhibition of Cyclic Nucleotide–gated Channels by Tyrosine Kinase Induced by Genistein

Rod photoreceptor cyclic nucleotide–gated (CNG) channels are modulated by tyrosine phosphorylation. Rod CNG channels expressed in Xenopus oocytes are associated with constitutively active protein tyrosine kinases (PTKs) and protein tyrosine phosphatases that decrease and increase, respectively, the apparent affinity of the channels for cGMP. Here, we examine the effects of genistein, a competitive inhibitor of the ATP binding site, on PTKs. Like other PTK inhibitors (lavendustin A and erbstatin), cytoplasmic application of genistein prevents changes in the cGMP sensitivity that are attributable to tyrosine phosphorylation of the CNG channels. However, unlike these other inhibitors, genistein also slows the activation kinetics and reduces the maximal current through CNG channels at saturating cGMP. These effects occur in the absence of ATP, indicating that they do not involve inhibition of a phosphorylation event, but rather involve an allosteric effect of genistein on CNG channel gating. This could result from direct binding of genistein to the channel; however, the time course of inhibition is surprisingly slow (>30 s), raising the possibility that genistein exerts its effects indirectly. In support of this hypothesis, we find that ligands that selectively bind to PTKs without directly binding to the CNG channel can nonetheless decrease the effect of genistein. Thus, ATP and a nonhydrolyzable ATP derivative competitively inhibit the effect of genistein on the channel. Moreover, erbstatin, an inhibitor of PTKs, can noncompetitively inhibit the effect of genistein. Taken together, these results suggest that in addition to inhibiting tyrosine phosphorylation of the rod CNG channel catalyzed by PTKs, genistein triggers a noncatalytic interaction between the PTK and the channel that allosterically inhibits gating.     

Rod cyclic nucleotide-gated channels have a stoichiometry of three CNGA1 subunits and one CNGB1 subunit

Phototransduction relies on the precise balance of speed and sensitivity to achieve optimal performance. The cyclic nucleotide-gated (CNG) ion channels, with their Ca(2+) permeability, high sensitivity to changes in cytosolic cGMP, rapid gating kinetics, and Ca(2+)-calmodulin modulation, are beautifully optimized for their role in light detection. Many of these specializations come about from the heteromeric composition of the native channel, comprised of CNGA1 and CNGB1 subunits. However, the stoichiometry and arrangement of these subunits is unknown. Here we have used an approach based on fluorescence resonance energy transfer (FRET) to determine the composition of the intact functional channel in the surface membrane. We find, surprisingly, that the channel contains three CNGA1 subunits and only one CNGB1 subunit. These results have implications for CNG channel function in particular and assembly of membrane proteins in general.     

Mechanism involved in the modulation of photoreceptor-specific cyclic nucleotide- gated channel by the tyrosine kinase adapter protein Grb14

We recently found that growth factor receptor-bound (Grb) protein 14 is a novel physiological modulator of photoreceptor specific cyclic nucleotide-gated channel alpha subunit (CNGA1). Grb14 promotes the CNG channel closure through its Ras-associating (RA) domain. In the current study we show that this RA domain-mediated inhibition of rod CNG channel is electrostatic in nature. Grb14 competes with cGMP for the CNGA1 binding pocket and electrostatically interacts with Arg⁵⁵⁹ through a negatively charged β-turn at its RA domain. Moreover, the three Glu residues (180--182) in Grb14 are absolutely critical for electrostatic interaction with the cGMP binding pocket and resultant inhibition. Our study also demonstrates that substitution of Lys140 for Ala or in combination with polyglutamte mutants of Grb14 results in a significantly reduced binding with CNGA1. These results suggest that in addition to Glu^180--182 and Lys¹⁴⁰, other residues in Grb14 may be involved in the electrostatic interaction with CNGA1. The RA domain is highly conserved among the members of Grb7 family of proteins, which includes Grb7, Grb10 and Grb14. Further, only Grb14 is able to modulate the channel activity, but not Grb7 and Grb10. All together, it suggests the existence of a divergence in RA domains among the members of the Grb7 family.     

State-dependent block of CNG channels by dequalinium

Cyclic nucleotide-gated (CNG) ion channels are nonselective cation channels with a high permeability for Ca(2+). Not surprisingly, they are blocked by a number of Ca(2+) channel blockers including tetracaine, pimozide, and diltiazem. We studied the effects of dequalinium, an extracellular blocker of the small conductance Ca(2+)-activated K(+) channel. We previously noted that dequalinium is a high-affinity blocker of CNGA1 channels from the intracellular side, with little or no state dependence at 0 mV. Here we examined block by dequalinium at a broad range of voltages in both CNGA1 and CNGA2 channels. We found that dequalinium block was mildly state dependent for both channels, with the affinity for closed channels 3-5 times higher than that for open channels. Mutations in the S4-S5 linker did not alter the affinity of open channels for dequalinium, but increased the affinity of closed channels by 10-20-fold. The state-specific effect of these mutations raises the question of whether/how the S4-S5 linker alters the binding of a blocker within the ion permeation pathway.